The assembly and disassembly of F-actin polymers from G-actin monomers is both spatially and temporally regulated in eukaryotic cells. This regulation is vital to cell motility, endocytosis, and cell division. We have continued our in vitro studies on actin polymerization and its regulation by purified proteins. Rate constants have been determined for the polymerization process by quantitative analysis of assembly progress curves and the use of chemically cross-linked actin trimers as seeds for assembly. Comparison of ADP-actin and ATP-actin has shown that nucleotide hydrolysis is not required for assembly, but that the rate constants for assembly are higher, and for disassembly are smaller, when ATP is present.